Objectives

1) To develop an understanding about the transformation.

2) Transform bacteria cells with a foreign DNA.

Theory

DNA can be exchanged among bacteria by three methods: transformation, transduction and conjugation. Transformation is one of the most popular techniques of molecular genetics because it is often the best way to reintroduce experimentally altered DNA into cells. This technique was first discovered in bacteria, but other ways have been designed to transform many types of animal and plant cell as well. Transformation was the first mechanism of bacterial gene exchange to be discovered. The initial experiment on transformation was performed by Frederick Griffith in England in 1928 when he was working with two strains of Streptococcus pneumoniae. Griffith’s experiments were based on the fact that S.pneumoniae makes two types of different appearing colonies, one type made by pathogenic bacteria and the other type made by bacteria that are incapable of causing infections. The colonies made by the pathogenic strains appear smooth on agar plates, because the bacteria excrete a polysaccharide capsule. Bacterial transformation has been widely investigated in Pneumococcus, Haemophilus influenzae, Bacillus subtilis, and certain other bacteria. During the process of transformation, genes are transferred from one bacterium to another as ‘naked’ DNA solution. In nature, some bacteria, perhaps after death and cell lysis, release their DNA into the environment. Other bacteria can then encounter the DNA and, depending on the particular species and growth conditions, take up fragments of DNA and integrate them into their own chromosomes by recombination. Transformation works best when the donor and recipient cells are very closely related.

Two elements are required in a transformation system. The first element is a suitable host bacterium. For this, commonly we use E.coli as host organism. The strain of E.coli has been cultured in the laboratory and it has been selected for characteristics that make it especially useful in the molecular biology laboratory. Plasmid is the other important element in the transformation system. Plasmid encodes some enzymes and antibiotic resistant markers which are expressed in the bacterium after transformation. When transformation occurs, the DNA transferred is often a plasmid: small, circular DNA found naturally in many bacteria. Plasmid is found as extra chromosomal DNA and it contains some genes that the bacterium would not normally possess. These extra genes can provide a growth advantage to bacteria by providing the gene for an enzyme such as amylase, beta- lactamase.

There are two forms of transformation: natural and artificial, each process depends on the ability of the organism to transform the DNA into the host cells. In natural transformation, bacteria are capable of DNA naturally which means they can take up DNA from their environment directly. That kind of bacteria is called as naturally transformable. In artificial transformation, bacteria are not naturally transformable which they do not take up DNA from the environment. Bacterial cells have been exposed to particular chemical or electrical treatments to make them more permeable and then only the cells can take up DNA efficiently. Bacteria can take up DNA artificially by using different techniques such as electroporation, heat shock, Ca2+ treatment of cells and protoplast uptake of DNA. In these techniques, cells made permeable to DNA by calcium ion treatment will take up both single stranded and double stranded DNA. Therefore, both linear and double stranded circular plasmids can be efficiently introduced into chemically treated cells. This fact has made calcium ion induced competence very useful for cloning and other applications that require the introduction of plasmid and phage DNA into cell.

Bacteria should possess requisite molecular characteristics to be susceptible for transformation- called competence. Transformation of naturally competent bacteria with plasmid or phage DNA usually occurs only with DNAs that are dimerized or multimerized into long concatemers. A dimerized or multimerized DNA is one in which two or more copies of the molecule are linked to head and tail. If a dimerized plasmid or phage DNA is cut only once, it still has complementary sequences at its ends that can recombine to recyclize the plasmid. The process of bacterial transformation and autonomous replication of the engineered plasmid DNA allows the production of large amounts of the DNA of interest within the bacterial host. This allows for further manipulations of the cloned DNA or for the expression of the gene of interest in the bacteria itself. The produced protein may result in a new bacterial phenotype, or the protein itself may be the desired end product.

Principle

Transformation process allows a bacterium to take up genes from its surrounding environment; that is transformation involves the direct uptakes of fragments of DNA by a recipient cell and the acquisition of new genetic characteristics. There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. The second major parameter is the genetic constitution of the host strain of the organism being transformed. Competent cells are capable of uptaking DNA from their environment and expressing DNA as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Calcium chloride treatment is one of the best methods for the preparation of competent cells. Competence results from alterations in the cell wall that makes it permeable to large DNA molecules. This is a naturally occurring process and through this bacteria can transfer advantageous characteristics, such as antibiotic resistance. Bacteria can take DNA from the environment in the form of plasmid. Most of them are double stranded circular DNA molecules and many can exist at very high copy numbers within a single bacterial cell. Many naturally occurring plasmids carry an antibiotic resistant gene referred to as a marker.

In the process of transformation, the competent cells are incubated with DNA in ice. Then it is placed in a water bath at 42ºC and further plunging them in ice. This process will take up the DNA into the bacterial cell. Then it is plated in an agar plate containing appropriate antibiotic. The presence of an antibiotic marker on the plasmid allows for rapid screening of successful transformants. Blue –white selection (Alpha complementation) can be used to determine which plasmids carry an inserted fragment of DNA and which do not. These plasmids contain an additional gene (lac Z) that encodes for a portion of the enzyme β – galactosidase. When it transformed into an appropriate host, one containing the gene for the remaining portion of β –galactosidase, the intact enzyme can be produced and these bacteria form blue colonies in the presence of X – gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside) and a gratuitous inducer called IPTG (Isopropyl β-D- Thiogalactopyronoside). These plasmids contains a number of cloning sites within the lac Z gene, and any insertion of foreign DNA into this region results in the loss of the ability to form active β –galactosidase. Therefore colonies that carry the plasmid with the insert, ie, Transformants will remain white and the colonies without the foreign DNA (Non-Transformants) will remain Blue. We can also calculate the efficiency of transformation by using the concentration of DNA and number of transformed colonies.